PaCas9 nuclease

ABSTRACT

The present invention relates to the field of biotechnology, molecular biology and medicine, in particular to nuclease enzyme and use thereof. More specifically, the present invention relates to PaCas9 nuclease enzyme. The invention also relates to a nucleic acid encoding said nuclease, a genetic construct, an expression vector, a delivery vector, which comprise said nucleic acid, a liposome comprising said nuclease or nucleic acid encoding said nuclease, a method for producing a nuclease, methods for delivery, and a host cell comprising a nucleic acid encoding said nuclease.

FIELD OF THE INVENTION

The present invention relates to the field of biotechnology, molecularbiology and medicine, in particular to nuclease enzyme and use thereof.More specifically, the present invention relates to PaCas9 nucleaseenzyme. The invention also relates to a nucleic acid encoding saidnuclease, a genetic construct, an expression vector, a delivery vector,which comprise said nucleic acid, a liposome comprising said nuclease ornucleic acid encoding said nuclease, a method for producing a nuclease,methods for delivery, and a host cell comprising a nucleic acid encodingsaid nuclease.

BACKGROUND

In 2007, it was first shown that CRISPR-Cas is an adaptive immune systemin many bacteria and most of archaea (Barrangou et al., 2007, Science315: 17091712, Brouns et al., 2008, Science 321: 960-964). Based onfunctional and structural criteria, three types of CRISPR-Cas systemshave so far been characterized, most of which use small RNA molecules asguide to target complementary DNA sequences (Makarova et al., 2011, NatRev Microbiol 9: 467-477, Van der Oost et al., 2014, Nat Rev Microbiol12: 479-492).

In a recent study by the Doudna/Charpentier labs, a thoroughcharacterization of the effector enzyme of the type II CRISPR-Cas system(Cas9) was performed, including demonstration that the introduction ofdesigned CRISPR RNA guides (with specific spacer sequences) targetscomplementary sequences (protospacers) on a plasmid, causing doublestrand breaks of this plasmid (Jinek et al., 2012, Science 337:816-821). Later, Jinek et al., 2012 used Cas9 as a tool for genomeediting.

Cas9 has been used to engineer the genomes of a range of eukaryoticcells (e.g. fish, plant, man) (Charpentier and Doudna, 2013, Nature 495:50-51).

Moreover, Cas9 has been used to improve yields of homologousrecombination in bacteria by selecting for dedicated recombinationevents (Jiang et al., 2013, Nature Biotechnol 31: 233-239). To achievethis, a toxic fragment (targeting construct) is co-transfected with arescuing fragment carrying the desired alteration (editing construct,carrying point mutation or deletions). The targeting construct consistsof Cas 9 in combination with a design CRISPR and an antibioticresistance marker, defining the site of the desired recombination on thehost chromosome; in the presence of the corresponding antibiotic,integration of the targeting construct in the host chromosome isselected. Only when the additional recombination occurs of the editingconstruct with the CRISPR target site elsewhere on the host chromosome,the host can escape from the auto-immunity problem. Hence, in thepresence of the antibiotic, only the desired (marker-free) mutants areable to survive and grow. A related strategy to select for subsequentremoval of the integrated targeting construct from the chromosome ispresented as well, generating a genuine marker-free mutant.

It has been established in recent years that CRISPR-Cas-mediated genomeediting constitutes a useful tool for genetic engineering. It has beenestablished that the prokaryotic CRISPR systems serve their hosts asadaptive immune systems (Jinek et al., 2012, Science 337: 816-821) andcan be used for quick and effective genetic engineering (for example,Mali et al., 2013, Nat Methods 10: 957-963), requiring only modificationof the guide sequence in order to target sequences of interest.

However, there is a continuing need for the development of agents withimproved sequence-specific nucleic acid detection, cleavage andmanipulation under a variety of experimental conditions for applicationin the area of genetic research and genome editing.

BRIEF SUMMARY OF INVENTION

The present invention relates to PaCas9 nuclease having an amino acidsequence of SEQ ID NO: 2.

In one aspect, the present invention relates to an isolated nucleic acidmolecule encoding PaCas9 nuclease having the nucleotide sequence of SEQID NO: 1.

In one aspect, the present invention relates to an expression vectorcomprising nucleic acid having the nucleotide sequence of SEQ ID NO: 1.

In some embodiments, the expression vector is a genetic construct asshown in FIG. 1, PpCas9-T2A-GFP-sgRNA1-MCS-sgRNA2-MCS.

In one aspect, the present invention relates to a vector to deliver atherapeutic agent comprising nucleic acid having the nucleotide sequenceof SEQ ID NO: 1.

In one embodiment of the present invention, the vector delivers thetherapeutic agent to target cells or target tissues.

In one aspect, the present invention relates to a liposome to deliver atherapeutic agent comprising PaCas9 nuclease having an amino acidsequence of SEQ ID NO: 2 nucleic acid having the nucleotide sequence ofSEQ ID NO: 1.

In one embodiment of the present invention, the liposome delivers thetherapeutic agent to target cells or target tissues.

In one aspect, the present invention relates to a method for deliveringa therapeutic agent to target cells or target tissues using the abovevector or the above liposome.

In one embodiment of the method, the therapeutic agent is delivered tothe target cells or target tissues by way of administering the abovevector or the above liposome into a mammalian body.

In one aspect, the present invention relates to a method for producing ahost cell to produce PaCas9 nuclease having an amino acid sequence ofSEQ ID NO: 2, which includes transformation of the cell using any of theabove vector.

In one aspect, the present invention relates to a method for producingthe PaCas9 nuclease, which comprises culturing the above host cell in agrowth medium under conditions sufficient to produce said PaCas9nuclease, if necessary, followed by isolation and purification of theobtained PaCas9 nuclease.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 Circular scheme of plasmid PpCas9-T2A-GFP-sgRNA1-MCS-sgRNA2-MCSintended for production of PpCas9 nuclease in mammalian cells.

AmpR is a beta-lactamase gene that provides resistance to ampicillin,

CMV promoter is the promoter of cytomegalovirus early genes,

Kozak sequence is intended to enhance the translation efficiency ofprotein,

START codon is a start codon,

NLS refers to nuclear localization signals (NLS),

PaCas9 is a nucleotide sequence of SEQ ID NO: 1 encoding PaCas9 nucleasehaving an amino acid sequence of SEQ ID NO: 2,

FLAG is a FLAG epitope sequence used for protein detection,

GFP is the modified green fluorescent protein,

TK pA refers to a thymidine kinase poly-A signal sequence used toincrease mRNA stability

F1 ori is an origin of replication which allows packaging of phagemidinto phage particles when cotransformed with helper phages,

polIII term+U6 promotor refers to cassettes for the expression of smallRNA molecules, each cassette contains U6 promoter and RNA polymerase IIItranscription terminator.

pUC origin is pUC replication origin in bacteria.

FIG. 2. Amino acid sequence of PaCas9 nuclease with domain distribution.

DESCRIPTION OF THE INVENTION

Definitions and General Methods

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art.

Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.Typically, the classification and methods of cell culture, molecularbiology, immunology, microbiology, genetics, analytical chemistry,organic synthesis chemistry, medical and pharmaceutical chemistry, aswell as hybridization and chemistry of protein and nucleic acidsdescribed herein are well known and widely used by those skilled in theart. Enzyme reactions and purification methods are performed accordingto the manufacturer's instructions, as is common in the art, or asdescribed herein.

A “mammal” refers to any animal that is classified as a mammal,including primates, humans, rodents, dogs, cats, cattle, small cattle,horses, pigs, etc.

Nuclease

Nucleases are a broad group of enzymes that hydrolyze the phosphodiesterbonds between nucleic acid subunits.

Depending on their specificity and activity, nucleases can be of thefollowing types: exonucleases and endonucleases, ribonucleases anddeoxyribonucleases, restrictases and some others. Restrictases are animportant element in applied molecular biology.

PaCas9 nuclease relates to the type of deoxyribonucleases.

PaCas9 nuclease is capable of cleaving DNA comprising a target nucleicacid sequence, when binding to at least one RNA molecule that recognizesthe target sequence.

PaCas9 nuclease comprises two endonuclease domains, which, one by one,make single-strand breaks, and, when acting together, make adouble-strand break.

PaCas9 nuclease is an effector enzyme of the type II CRISPR-Cas system(nuclease of type 2).

PaCas9 nuclease is capable of making a double-strand DNA break with ahighly specific recognition site (16-20 letters).

DNA of PaCas9 nuclease is presented in SEQ ID NO:1.

Amino acid sequence of PaCas9 nuclease is presented in SEQ ID NO:2.

FIG. 2 shows an amino acid sequence of PaCas9 nuclease with domaindistribution.

PaCas9 nuclease is associated with clustered regularly interspaced shortpalindromic repeats (CRISPR), as well as other adjacent components ofthe CRISPR-Cas system: crRNA and tracrRNA sequences.

A nucleotide sequence encoding tracrRNA is presented in SEQ ID NO:3.

A nucleotide sequence encoding a direct repeat DR is presented in SEQ IDNO:4.

crRNA consists of a target-dependent variable part and a direct repeatDR presented in SEQ ID NO:4.

The term “therapeutic agent” herein refers to PaCas9 nuclease having anamino acid sequence of SEQ ID NO: 2 or to an isolated nucleic acidmolecule encoding the PaCas9 nuclease and having a nucleotide sequenceof SEQ ID NO: 1.

tracrRNA (trans-activating crRNA) is a small trans-encoded RNA.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) arespecial bacterial and archaeal loci consisting of direct repeats thatare interspaced with unique sequences (spacers).

Nucleic Acid Molecules

The terms “nucleic acid”, “nucleic sequence”, “nucleic acid sequence”,“polynucleotide”, “oligonucleotide”, “polynucleotide sequence” and“nucleotide sequence”, used interchangeably in the present description,mean a precise sequence of nucleotides, modified or not, determining afragment or a region of a nucleic acid, containing unnatural nucleotidesor not, and being either a double-strand DNA or RNA, a single-strandedDNA or RNA, or transcription products of said DNAs.

It should also be included here that the present invention does notrelate to nucleotide sequences in their natural chromosomal environment,i.e., in a natural state. The sequences of the present invention havebeen isolated and/or purified, i.e., they were sampled directly orindirectly, for example by a copy, their environment having been atleast partially modified. Thus, isolated nucleic acids obtained byrecombinant genetics, by means, for example, of host cells, or obtainedby chemical synthesis should also be mentioned here.

An “isolated” nucleic acid molecule is one which is identified andseparated from at least one nucleic acid molecule-impurity, which theformer is bound to in the natural source of nuclease nucleic acid. Anisolated nucleic acid molecule is different from the form or set inwhich it is found under natural conditions. Thus, an isolated nucleicacid molecule is different from a nucleic acid molecule that exists incells under natural conditions. An isolated nucleic acid moleculehowever includes a nucleic acid molecule located in cells in which theantibody is normally expressed, for example, if the nucleic acidmolecule has a chromosomal localization that is different from itslocalization in cells under natural conditions.

The term “nucleotide sequence” encompasses the complement thereof unlessotherwise specified. Thus, a nucleic acid having a particular sequenceshould be understood as one which encompasses the complementary strandthereof with the complementary sequence thereof.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader sequence is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; aribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous.

Vector

The term “vector” as used herein means a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked. Insome embodiments, a vector is a plasmid, i.e., a circular double-strandpiece of DNA into which additional DNA segments may be ligated. In someembodiments, a vector is a viral vector, wherein additional DNA segmentsmay be ligated into the viral genome. In some embodiments, vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin site ofreplication and episomal mammalian vectors). In further embodiments,vectors (e.g., non-episomal mammalian vectors) can be integrated intothe genome of a host cell upon introduction into a host cell, andthereby are replicated along with the host gene. Moreover, certainvectors are capable of directing the expression of genes to which theyare operatively linked. Such vectors are referred to herein as“recombinant expression vectors” (or simply, “expression vectors”).

In one aspect, the present invention relates to a vector suitable forthe expression of any of nucleotide sequences described herein.

The present invention relates to vectors comprising nucleic acidmolecules that encode PaCas9 nuclease.

In some embodiments, the PaCas9 nuclease of the invention is expressedby inserting DNA into expression vectors, so that the genes arefunctionally linked to the necessary expression control sequences, suchas transcriptional and translational control sequences. Expressionvectors include plasmids, retroviruses, adenoviruses, adeno-associatedviruses (AAV), plant viruses, such as cauliflower mosaic virus, tobaccomosaic virus, cosmids, YACs, EBV derived episomes, and the like. DNAmolecules may be ligated into a vector such that transcriptional andtranslational control sequences within the vector serve their intendedfunction of regulating the transcription and translation of the DNA. Anexpression vector and expression control sequences may be chosen to becompatible with the expression host cell used. DNA molecules can beintroduced into an expression vector by standard methods (e.g., ligationof complementary restriction sites on an PaCas9 nuclease gene fragmentand vector, or blunt end ligation if no restriction sites are present).

In addition to the PaCas9 nuclease gene, the recombinant vectorexpression of the invention can carry regulatory sequences that controlthe expression of the PaCas9 nuclease gene in a host cell. It will beunderstood by those skilled in the art that the design of an expressionvector, including the selection of regulatory sequences, may depend onsuch factors as the choice of a host cell to be transformed, the levelof expression of a desired protein, and so forth. Preferred controlsequences for an expression host cell in mammals include viral elementsthat ensure high levels of protein expression in mammalian cells, suchas promoters and/or enhancers derived from a retroviral LTR,cytomegalovirus (CMV) (such as a CMV promoter/enhancer), simian virus 40(SV40) (such as a SV40 promoter/enhancer), adenovirus, (e.g., the majorlate promoter adenovirus (AdMLP)), polyomavirus and strong mammalianpromoters such as native immunoglobulin promoter or actin promoter. Forfurther description of viral control elements and sequences thereof,see, e.g., U.S. Pat. Nos. 5,168,062, 4,510,245 and 4,968,615. Methodsfor expressing polypeptides in bacterial cells or fungal cells, e.g.,yeast cells, are also well known in the art.

In addition to the PaCas9 nuclease gene and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of a vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates the selection of host cells intowhich a vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216,4,634,665 and 5,179,017). For example, typically the selectable markergene confers resistance to medicinal agents, such as G418, hygromycin ormethotrexate, on a host cell into which a vector has been introduced.For example, selectable marker genes include a dihydrofolate reductase(DHFR) gene (for use in dhfr-host cells during methotrexateselection/amplification), a neo gene (for G418 selection), and aglutamate synthetase gene.

The term “expression control sequence” as used herein is intended torefer to polynucleotide sequences that are necessary to effect theexpression and processing of coding sequences to which they are ligated.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include the promoter of ribosome binding site, andtranscription termination sequences; in eukaryotes, typically, suchcontrol sequences include promoters and transcription terminationsequences. The term “control sequences” is intended to include at leastall components, the presence of which is essential for expression andprocessing, and can also include additional components, the presence ofwhich is advantageous, for example, leader sequences and fusion partnersequences.

Host Cells

The term “recombinant host cell” (or simply “host cell”) as used hereinis intended to refer to a cell into which a recombinant expressionvector has been introduced. The present invention relates to host cells,which may include, for example, a vector according to the inventiondescribed above. It should be understood that “recombinant host cell”and “host cell” are intended to refer not only to a particular subjectcell but to the progeny of such a cell as well. Since modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to a parentalcell, however, such cells are still included within the scope of theterm “host cell” as used herein.

Nucleic acid molecules encoding PaCas9 nuclease of the invention andvectors comprising these nucleic acid molecules can be used fortransfection of a suitable mammalian or cell thereof, plant or cellthereof, bacterial or yeast host cell. Transformation can be by anyknown technique for introducing polynucleotides into a host-cell.Methods for introduction of heterologous polynucleotides into mammaliancells are well known in the art and include dextran—mediatedtransfection, cationic polymer-nucleic acid complex transfection,calcium phosphate precipitation, polybrene—mediated transfection,protoplast fusion, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of DNA into nuclei. In addition, nucleic acidmolecules may be introduced into mammalian cells by viral vectors.Methods for transfecting cells are well known in the art. See, e.g.,U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461 and 4,959,455. Methodsfor transforming plant cells are well known in the art, including, e.g.,Agrobacterium-mediated transformation, biolistic transformation, directinjection, electroporation and viral transformation. Methods oftransforming bacterial and yeast cells are also well known in the art.

Mammalian cell lines used as hosts for transformation are well known inthe art and include a plurality of immortalized cell lines available.These include, e.g., Chinese hamster ovary (CHO) cells, NS0 cells, SP2cells, HEK-293T cells, FreeStyle 293 cells (Invitrogen), NIH-3T3 cells,HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidneycells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549cells, and a number of other cell lines. Cell lines are selected bydetermining which cell lines have high expression levels and provide fornecessary characteristics of protein produced. Other cell lines that maybe used are insect cell lines, such as Sf9 or Sf21 cells. Whenrecombinant expression vectors encoding the PaCas9 nuclease areintroduced into mammalian host cells, the PaCas9 nuclease are producedby culturing the host cells for a period of time sufficient to allow forexpression of the PaCas9 nuclease in host cells or, more preferably,secretion of the PaCas9 nuclease into the culture medium in which thehost cells are grown. The PaCas9 nuclease can be isolated from theculture medium using standard protein purification techniques. Planthost cells include, e.g., Nicotiana, Arabidopsis, duckweed, corn, wheat,potato, etc. Bacterial host cells include Escherichia and Streptomycesspecies. Yeast host cells include Schizosaccharomyces pombe,Saccharomyces cerevisiae and Pichia pastoris.

Furthermore, level of production of the PaCas9 nuclease of the inventionfrom production cell lines can be enhanced using a number of knowntechniques. For example, the glutamine synthetase gene expression system(the GS system) is a common approach for enhancing expression undercertain conditions. The GS system is discussed in whole or part inconnection with EP Nos. 0216846, 0256055, 0323997 and 0338841.

It is likely that the PaCas9 nuclease obtained from different cell linesor from transgenic animals will have a different glycosylation profileas compared to each other. However, the PaCas9 nuclease encoded by thenucleic acid molecules described herein is part of the presentinvention, regardless of the glycosylation state, and, in general,regardless of the presence or absence of post-translationalmodifications.

Liposome

In one aspect, the present invention relates to liposomes encapsulatingthe PaCas9 nuclease having an amino acid sequence of SEQ ID NO: 2 or toan isolated nucleic acid molecule encoding the PaCas9 nuclease andhaving a nucleotide sequence of SEQ ID NO: 1.

Liposomes are microscopic closed vesicles having an internal phase,surrounded by one or more lipid bilayers, and ability to holdwater-soluble material in the internal phase, and oil-soluble materialin the phospholipid bilayer. When entrapping an active compound inliposome, and delivering it to target tissue, how to entrap the activecompound in the liposome with high efficiency, and how to secure stableretention of the active compound by the liposome constitute importantissues.

In general, a liposome is considered a particle with a predominant sizeof several tens of nanometers up to tenths of a micron, its shellaccommodating molecules of another substance(s). The liposome shell is“semi-permeable” to water molecules and ions.

Liposomes are characterized by the ability to contain and retainsubstances of different nature. The range of substances incorporated inliposomes is quite wide, ranging from inorganic ions andlow-molecular-weight organic compounds to large proteins and nucleicacids.

Liposomes provide a prolonged release of a substance incorporated in acarrier.

Liposomes can be made from phospholipid, in particular fromphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidylglycerol, phosphatidic acid,sphingophospholipid, egg/soybean phospholipids or mixtures thereof.

EXAMPLES

The following examples are provided for better understanding of theinvention. These examples are for purposes of illustration only and arenot to be construed as limiting the scope of the invention in anymanner.

All publications, patents, and patent applications cited in thisspecification are incorporated herein by reference. Although theforegoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, itwill be readily apparent to those of ordinary skill in the art in lightof the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended embodiments.

Materials and General Methods

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al, Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York, 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene Synthesis

Desired gene segments were prepared from oligonucleotides made bychemical synthesis. The gene segments of 300-4000 kb long, which wereflanked by singular restriction sites, were assembled by annealing andligation of oligonucleotides including PCR amplification andsubsequently cloned via the indicated restriction sites. The DNAsequences of the subcloned gene fragments were confirmed by DNAsequencing.

DNA Sequence Determination

DNA sequences were determined by Sanger sequencing.

DNA and Protein Sequence Analysis and Sequence Data Management

The Infomax's Vector NTI Advance suite version 8.0 was used for sequencecreation, mapping, analysis, annotation and illustration.

Expression Vectors

For the expression of the PaCas9 nuclease, variants of expressionplasmids intended for expression in prokaryotic cells (E.coli),transient expression in eukaryotic cells (e.g., in CHO cells) were used.Beside the PaCas9 nuclease expression cassette the vectors contained: anorigin of replication which allows replication of said plasmid in E.coli, genes which confer resistance in E. coli to various antibiotics(e.g., to ampicillin and/or kanamycin).

Example 1 Method of Preparation of PaCas9 Nuclease

To prepare metagenomic sequences, samples of Homoeodictya palmatasponges were collected from regions of the White Sea, the material wasfractionated by centrifugation, then total DNA was isolated andsubsequently sequenced.

An open reading frame of the PaCas9 protein, as well as adjacentcomponents of CRISPR-Cas system (CRISPR cassette, as well as crRNA andtracrRNA sequences) were detected in the metagenomic sequences usingbioinformatics methods.

DNA of PaCas9 nuclease is presented in SEQ ID NO:1.

The amino acid sequence of PaCas9 nuclease is presented in SEQ ID NO:2.

A nucleotide sequence encoding tracrRNA is presented in SEQ ID NO:3.

A nucleotide sequence encoding a direct repeat DR is presented in SEQ IDNO:4.

Example 2 Description of Cloning

The PaCas9 nuclease gene sequence was obtained by way of bioinformaticsearch. The sequence was codon-optimized to ensure optimal expression inmammalian cells, and then assembled de novo from chemically synthesizedoligonucleotides using the Gibson method. The synthesized PaCas9 genewas cloned in a genetic construct from the 3′-end of the CMV promoter.Kozak sequences and nuclear localization signals (NLS) were added fromthe 5′-end of the gene, and FLAG epitope sequence for protein detectionwas added from the 3′-end. After the PaCas9 sequence and its associatedelements as listed above, T2A elements and the open reading frame of thegreen fluorescent protein (EGFP) as a marker for expression are placedin the construct in the same reading frame.

After the reading frames, a thymidine kinase poly-A signal sequence isplaced from the 3′-end to increase the stability of mRNA. There are twocassettes in tandem in the area of the bacterial cortex of the geneticconstruct for expression of small RNA molecules. Each cassette containsa U6 promoter and RNA polymerase III transcription terminator. Thesecassettes are necessary for the expression of RNA molecules that providespecific interaction of the PaCas9 protein with the target DNA molecule(cellular genome). The construct map is shown in FIG. 1. This constructallows for expressing both the PaCas9 protein (which is transported tothe nucleus through NLS) and RNA molecules guiding the protein (guidingRNAs), as well as detecting the protein by FLAG epitope and determiningthe efficiency of delivery of the genetic construct by detection ofEGFP.

Example 3 Enzymatic Activity of PaCas9 Protein

Amino acids involved in enzymatic hydrolysis of DNA/RNA were identifiedby comparing the homology of HNH and RuvC domains of various Cas9 familyproteins with PaCas9 domains (domain distribution is shown in FIG. 2).Conservative amino acids, for which participation in the enzymaticactivity of Cas9 proteins was previously shown, were isolated in PaCas9.Thus, it was found by analytical methods that the amino acid residues ofthis protein are necessary for the enzymatic activity of PaCas9 protein(amino acid—position): D 9; E 527; H 750; D 753; H 613; N 636.

Example 4 Determination of Enzymatic Activity of PaCas9 Protein

To determine PAM (Protospacer Adjacent Motif) sequence, we performed invitro reactions of cutting DNA libraries using a recombinant nucleaseprotein (SEQ ID NO: 2), crRNA (consists of a target-dependent variablepart and a direct repeat presented in SEQ ID NO:4) and tracrRNA (SEQ IDNO:3). DNA library is a PCR fragment comprising a seven-letterrandomized sequence, and a recognizable sequence, a protospacer.

After incubation of PaCas9-RNA-protein complex with the DNA library, thereaction products are loaded into gel electrophoresis. Uncut fragmentsare extracted from the gel and sequenced on Illumina platform.Comparison of PAM sequences contained in uncut PaCas9 reaction productsand a control reaction will allow to determine PAM of protein inquestion.

After identifying the PAM sequence, in vitro nuclease activity isevaluated. To this end, protein in complex with RNA guides is incubatedwith a DNA fragment carrying a protospacer sequence and identified PAM.Optimal ratio of the RNA-protein complex to cut DNA was determined.Nuclease activity was evaluated based on the amount of PaCas9 protein,required for 50% cutting of 200 ng of target DNA of about 400 base pairslong, containing the optimal PAM.

Thus, it was confirmed that PaCas9 nuclease has enzymatic activity andmakes a double-strand break in DNA.

Moreover, it was confirmed that PaCas9 nuclease is able to make adouble-strand break in DNA with a highly specific recognition site(16-20 letters).

1. PaCas9 nuclease having an amino acid sequence of SEQ ID NO:
 2. 2. Anisolated nucleic acid molecule encoding a PaCas9 nuclease according toclaim 1 and having a nucleotide sequence of SEQ ID NO:
 1. 3. Anexpression vector comprising a nucleic acid according to claim
 2. 4. Anexpression vector according to claim 3, which is a genetic constructshown in FIG.
 1. 5. A vector for delivering a therapeutic agentcomprising a nucleic acid according to claim
 2. 6. A vector according toclaim 5, wherein the therapeutic agent is delivered to target cells ortarget tissues.
 7. A method for delivery of a therapeutic agent to atarget cell or target tissue using a vector according to claim 3-6.
 8. Amethod for producing a host cell for producing a PaCas9 nucleaseaccording to claim 1, comprising transforming the cell with a vectoraccording to any one of claims 3-6.
 9. A host cell for producing aPaCas9 nuclease according to claim 1, comprising a nucleic acidaccording to claim
 2. 10. A method for producing a PaCas9 nucleaseaccording to claim 1, comprising culturing a host cell according toclaim 9 in a growth medium under conditions sufficient to produce saidPaCas9 nuclease, if necessary, followed by isolation and purification ofthe obtained PaCas9 nuclease.